JPH0157956B2 - - Google Patents

Abstract

A process is described for the production of fructose from glucose. An aqueous solution of glucose is converted to D-glucosone by an enzymatic process. D-glucosone is then converted to substantially pure fructose by chemical hydrogenation. Fructose may be recovered in crystalline form.

Description

[Detailed description of the invention]

The present invention generally relates to a method for producing D-glucosone, an intermediate for fructose production, from glucose by a one-step process, enzymatic oxidation. Various significant problems have been encountered in the commercial scale production of crystalline fructose. One of the biggest problems is manufacturing costs. The only commercially viable production method comparable to the current invention is to first produce a glucose-fructose mixture from glucose (using glucose isomerase or by performing alkaline isomerization) or sucrose (via invert sugar). producing syrup, then physically separating the two sugars (by ion exchange or selective calcium salt precipitation) and finally converting the aqueous solution into crystalline form (by seeding or methanol precipitation) ¹ fructose It is based on the process of collecting. Physical separation of the two sugars is required. This is because crystalline fructose is most difficult to recover from aqueous solutions ¹⁵ and often impossible if substantially all ionic materials, residual glucose and other contaminants are not removed. Such processing is expensive and therefore the market price for fructose is high and cannot compete with sugar cubes as a single serving. According to the literature, fructose can be made from glucosone in low concentrations. As early as 1889, fructose was produced by reducing D-glucosone with zinc dust in aqueous acetic acid. ⁶
This reduction has been performed as an analytical test for the detection of D-glucosone in various biological materials. ^8,10,12,
Reduction of D-glucosone to D-fructose is also accomplished with sodium borohydride. D
−Conversion of glucosone to fructose−
Since the reductase that performs CHO → CH ₂ O was known, it has become a viable enzymatic reaction. The literature also states that glucosone can be made from glucose in low concentrations by oxidizing glucose. Various methods have been reported, including those performed with H ₂ O ₂ ⁴ or copper acetate ⁵ . Each of these reactions has low conversion yields (less than 30%) and many by-products. Converting glucose to D-glucosone involves first producing a derivative of glucose (e.g., reacting with phenylhydrazine to form ^glucosazone6 or reacting with paratoluidine7 ⁾ and then chemically treating the derivative. Therefore, the method of manufacturing D-glucosone was used. These reactions did not give yields above 50%, and the reagents were not recoverable, making them too expensive for commercial purposes. In addition, the use of reagents containing aromatic compounds poses various problems in obtaining food-quality sugars. Enzymatic reactions are also known for converting glucose to D-glucosone. In 1932, glucose was
- a type of mollusc that oxidizes to glucosone,
Saxidomus giganteus
There is a description using a crystalline substance called giganteous) ⁸ . In 1937, the production of D-glucosone was developed by combining glucose, starch, maltose or sucrose with two molds, Aspargillus parasiticus and Aspergillus flauus oryzae.
oryzae) was reported to be carried out by oxidizing the plasma dissociated product. In 1956, enzymatic oxidation of glucose to glucosone was discovered in the red algae Iridophycus
flaccidum) ¹⁰ . A carbohydrate oxidase that oxidizes glucose to D-glucosone was isolated from the mycelium of the basidiomycete Polyporus obtusus.
No mention is made of yield. At the end, 1978
In 2007, glucose-2-oxidase activity was significantly increased in the basidiomycete, Oudemansiella
mucida), as well as ¹² other wood rot fungi. The top yield was not higher than 50%. In all of these cases, the production of D-glucosone is believed to be accompanied by the evolution of hydrogen peroxide. The diagram below is a process diagram centered on D-glucosone of the present invention. Propylene haloperoxidase――――――――――――→ Halide Propylene halohydrin Halohydrin――――――――→ Epoxidase Propylene oxide (hydrogen peroxide) Glucose carbohydrate―――― ---> Oxidase D-glucosone Chemical ---> Reduced fructose The formula below represents the molecular structure of D-glucose. The formula below represents the molecular structural formula of D-glucosone. The formula below represents the molecular structural formula of D-fructose. Regarding the molecular structure of D-glucosone, it will be recognized by those skilled in the art that several other molecular structures can be assumed to exist in aqueous solution. Many of these have cyclic structural formulas. ^{2
, 3} , the terms "glucose,""D-glucose," and "dextrose" are used interchangeably to encompass either the solution or dry form of this monosaccharide. The formula represents glucose. "D-glucosone" and "D-arabino-2-hexosulose" used here
The terms are used interchangeably. The formula represents D-glucosone. “Fructose” and “D-
The terms "fructose" and "levulose" are used interchangeably to refer to isomers of glucose that are sweeter than glucose. "Crystal fructose"
The term is used in application to include this monosaccharide in its anhydrous state. The formula represents fructose. Generally speaking, according to the present invention, glucose in an aqueous solution is enzymatically converted to D-glucosone with a suitable enzyme such as carbohydrate oxidase or glucose-2-oxidase.
This conversion proceeds spontaneously, rapidly and virtually completely. D-glucosone is not isolated beforehand and is converted to fructose by suitable chemical hydrogenation. This conversion also proceeds rapidly and essentially completely. The resulting fructose is recovered substantially free of glucose and all other sugars, either in aqueous solution or in solid form. Although the low-yield conversions of glucose to D-glucosone and D-glucosone to fructose mentioned and referenced above are known in the scientific literature, these two reactions producing fructose from glucose are The concept of combining has not been found in other literature to date. Additionally, the successful use of these two reactions to produce crystalline fructose from glucose in an economically and commercially useful process has not been reported prior to our invention. Neither the reports on the conversion of glucose to D-glucosone nor the conversion of D-glucosone to fructose considered the concept of producing crystalline fructose from glucose. D-glucosone was chemically synthesized because it was needed as a chemical standard. D-
Glucosone has been discovered in biological systems either unconsciously (e.g. as an unknown component in a biochemical pathway during research) or consciously (e.g. as part of a study of the biochemical oxidation of blood glucose). D-
Glucosone was reduced to fructose as a spot test for the detection of D-glucosone. The yield of D-glucosone obtained in the present invention exceeds the yield reported in the literature for this enzyme. Due to the high conversion rate to D-glucosone, there is no need to physically separate the remaining unconverted glucose. Some oxidoreductases are
It has a catalytic activity with respect to the oxidation of the hydroxyl group at the th carbon, that is, it promotes the oxidation of the hydroxyl group to a keto group, such as converting the structure of the formula to the structure of the formula. The specific oxidoreductase enzymes in the examples described herein refer to a variety of species such as "glucose-2-oxidase,""pyranose-2-oxidase," and "carbohydrate oxidase," but the present invention is not limited to the enzymes so indicated. Glucose-2-
Oxidase is highly specific for the substrate glucose, whereas carbohydrate oxidase
The enzyme has a broader substrate specificity, although glucose is a suitable substrate. Any enzyme capable of converting the hydroxyl group on the second carbon of glucose into a keto group without substantially affecting other groups on the glucose molecule is within the scope of our invention. Such an enzyme may be specified as a type of enzyme having glucose-2-oxidase activity. A suitable carbohydrate oxidase enzyme is derived from the microorganism Polyporus Obtusus. Glucose-2-oxidizer
The raw materials for zebra include several other microorganisms, the molluscs mentioned above, and red algae. These enzymes and their raw materials are merely shown and are not meant to include all suitable enzymes and their raw materials within the scope of the present invention. For ease of discussion, various aspects of the invention are characteristically, but not exclusively, characterized in that they include suitable carbohydrate oxidases of Polyporus obtusus and glucose-2-oxidases of Aspergillus oryzae. - will be mentioned in connection with the use of ze. Symptoms are collected according to the usual method.
Shake at room temperature and perform deep culture. Shake the enzyme;
It is produced from the mycelium of symptomatic organisms cultivated under deep culture conditions. Although this enzyme can be used as it is,
Rather, it is used in a fixed manner. Enzyme immobilization processes are well known to those skilled in the art and consist of reacting an enzyme solution with a portion of a wide range of surfaces of treated or untreated organic and inorganic supports.
Some of the carriers include polyacrylamide, ethylene maleic acid copolymer, methacrylic basic polymer, polypeptide, styrene-base polymer, polypeptide, styrene-base polymer, polypeptide, styrene-base polymer, These include agarose, cellulose, dextran, silica, porous glass beads, activated carbon or carbon black, wood and sawdust, hydroxyapatite, and aluminum or titanium hydroxide. This form of the enzyme increases stability, extends durability and availability, and regenerative capacity. Reactions carried out using immobilized enzymes can be passed through columns or carried out in reaction tanks or other suitable reactors. Carbohydrate oxidase or glucose-2
-In addition to the oxidase enzyme, enzyme raw materials are required. Additionally, methods for removing or utilizing hydrogen peroxide are necessary to most effectively carry out the conversion reaction of glucose to D-glucosone. This is because hydrogen peroxide oxidizes certain critical sites on the enzyme molecule, impairing its function. The process for removing hydrogen peroxide is (1) decomposition by the enzyme catalase (2)
(3) Decomposition by known chemical methods and (3) using a decomposition template, such as manganese oxide or carbon ^black , which is the same immobilized carrier for the oxidoreductase enzyme. Another suitable method is that rather than decomposing the hydrogen peroxide produced, it may be consumed to produce valuable by-products. As shown in the above scheme, combining the production of D-glucosone with the production of propylene halohydrin or propylene oxide is a preferred embodiment. The enzymatic conversion of glucose to D-glucosone is preferably carried out in an aqueous solution at approximately neutral pH;
can be performed within about 8 minutes. This conversion is suitably carried out at room temperature, but can be carried out at temperatures ranging from about 15°C to about 65°C. The appropriate pressure condition is atmospheric pressure, but it can also vary below or above atmospheric pressure. Any carbohydrate substance that produces glucose by chemical or enzymatic means is a suitable source of glucose for conversion to D-glucosone and fructose synthesis. These materials include, but are not limited to, cellulose, starch, sucrose, corn syrup, HFCS, and other syrups containing glucose and fructose in various ratios. The analytical methods used for the analysis of the sugars of the present invention are shown below: 1. Thin layer chromatography (TLC). Develop on an Avicel-coated glass plate with a solvent system with a volume ratio of isopropanol:pyridine:acetic acid:water of 8:8:2:4. glucose and fructose are
Rf0.5-0.6; D-glucosone shows streaks of Rf0.4-0.5. (Rf = distance traveled from the origin of the substance/distance from the origin to the tip of the solvent) Triphenyltetrazolium chloride (2
% TTC (0.5N sodium hydroxide solution), both D-glucosone and fructose produce immediate red spots. Glucose produces red spots only when heated at 100°C for 10 minutes. Diphenylamine/aniline/phosphoric acid/ethyl acetate reagent (0.15g/0.8ml/
When sprayed (11ml/100ml), glucose produces brown spots; D-glucosone produces purple streaks; and fructose produces brown spots at 95°C.
Produces yellow spots when heated for minutes. 2 High Performance Liquid Chromatography (HPLC) A.
μ-Bondapak - Carbohydrate column (purchased from Waters Associates)
Contains 0.001M potassium phosphate buffer (PH7)
Develop with a 15% acetonitrile aqueous solution at a flow rate of 2 ml/min. Glucose Rt11.5, D-glucosone Rt14.0, fructose Rt9.5 (Rt=
retention time). Analysis was performed using both a Waters Associates refractive index detector and a Schoeffel tunable ultraviolet detector (192 nm).
Spectral Physics SP8000 Instrument (Sp-ectra
Physics SP8000 instrument). 3 Mass spectrometry (MS). The following derivative manufacturing process is used to make the volatile chemical components: (a) 110 mg of N,N-diphenylhydrazine (H ₂ NNΦ ₂ ) and
Add 1 ml of 75% ethanol water. The reaction mixture is stirred and left at room temperature overnight. (b) Add 3 ml of water to the reaction mixture and separate the resulting precipitate from the supernatant by centrifugation and decantation. To this precipitate, pyridine-acetic anhydride 1:1
Add 1 ml of the mixture. The reaction mixture is left in a water bath at 35°-40°C for 15 minutes, shaking occasionally. (c) Add 2 ml of water to stop the reaction and then extract the mixture twice with 3 ml each of ethyl ether. The ether solution is dried with a small amount of anhydrous sodium sulfate, then the ether is gently heated (~40°C) and blown off with nitrogen. (d) Prepare the resulting solid or syrup for mass spectrometry. Expected reaction: -CHO -CH=NNO/ ₂ >C=O(1) -OH (2)H ₂ NNO/ ₂ -----------→ AC ₂ O, pyridine > C=O (unchanged substance ) -OAC Glucose produces a molecular ion in 556 squares (C ₂₈ H ₃₂ N ₂ O ₁₀ ) and a base ion in 168 squares (NO/ ₂ ion fragment); fructose produces a molecular ion in 390 squares (C ₁₆ H ₂₂ O ₁₁ ). D-glucosone produces molecular ions in 512 squares (C ₂₆ H ₂₈ N ₂ O ₉ ) and 223 squares (OC-CH=N-NO/ ₂ ion fragment)
Generates characteristic fragment ions that are resistant to The analysis was performed using a Finnigan gas chromatography/mass spectrometry/densitometer (GC/MS/DS) model 4023 instrument with electron impact ionization at 70 eV and a sample temperature of 220°. 4 Test tube colorimetric analysis. Analysis of D-glucosone in the presence of excess D-glucose is easily quantified using two colorimetric methods. There is a detection method based on the difference in the reduction rate of two sugars using triphenyltetrazolium chloride (TTC). Add 0.5 ml of sample, 0.1 ml of 1% TTC aqueous solution, and 0.4 ml of 6N NaOH into a 20 ml test tube. After exactly 5 minutes,
Add 15 ml of acetic acid:ethanol (1:9) and stir the tube contents. Using water as a blank test, using a Varian 635 ultraviolet/visible (UV/VIS) spectrometer,
Measure the absorbance at 480nm. glucose is
The reduction of TTC to red-triphenylformazane is approximately 100 times slower than the equivalent amount of D-glucosone. The detection method using diphenylamine/aniline/phosphoric acid reagents is based on the fact that different sugar structures produce different colors. Add 0.2 ml of sample and 5 ml of the following reagent mixture to a 20 ml test tube: Diphenylamine 0.15 g Aniline 0.80 ml Isopropanol 100 ml Phosphoric acid 11 ml Place the test tube in a 37° water bath for 60 minutes.
Glucose gives a yellow solution; D-glucosone gives a purple solution. The sources of pure sugars used in the various analytical aspects of the present invention are as follows: 1 D-glucose is available from Applied Science
Laboratories (Applied Science)
99% pure product purchased from Laboratories). 2 D-fructose is a 99% pure product purchased from Applied Science Laboratories. 3 D-glucosone was chemically synthesized by the following method: 20 g of glucose is mixed with 1 part of distilled water containing 27 ml of glacial acetic acid. phenylhydrazine
Add 44g. The reaction is continued at 80° C. for 3 hours with vigorous stirring using a stirrer and then cooled to room temperature overnight. Filter the solids and add with 10% acetic acid water.
Then wash with ethyl ether. Thoroughly dry the solid at 50°C in a vacuum oven. The experimental yield is 16.1 g glucosazone. Put this glucosazone in the three-necked flask from step 3 and add 450 ethanol.
ml, 750 ml of distilled water and 9 ml of glacial acetic acid.
Add 27.8 g of freshly made benzaldehyde and bring to reflux while stirring vigorously with a stirrer. Continue the reaction at reflux for 5 hours. Reverse the condenser and add distilled water (via addition funnel) to 750 mL
Distill 450 ml while adding ml to the flask.
The reaction is cooled overnight to precipitate the benzaldehyde phenylhydrazone. Filter this solution and wash the residue with distilled water (approx.
). Concentrate the liquid and washing liquid to 500 ml and extract 10 times with 300 ml of ethyl ether each.
Transfer to a rotary evaporator to remove remaining ethyl ether in the aqueous solution.
Distill for 30 minutes. This aqueous solution is strictly acetone-washed Amberlite XAD.
-2 through a 4 x 100 cm column.
The column is washed with an additional 200 ml of water to remove residual glucosone. The aqueous solution is collected and lyophilized. The experimental yield of glucosone is
3.4g (16% overall yield). The following examples illustrate various features of the invention, but are in no way meant to limit the scope of the invention as defined in the appended claims. Unless indicated otherwise, all temperatures are room temperature (approximately 25°C) and all pressures are atmospheric (approximately 1
It is atmospheric pressure. Example 1 Substantially complete conversion of glucose to D-glucosone using immobilized carbohydrate oxidase is demonstrated in this example. Glucose (2 g) is added to 20 ml of distilled water in a 100 ml Pyrex flask and the sugar solution is stirred. Oxygen gas was blown into the flask, and catalase [Sigma
Chemical Co.), C-40, manufactured from cow liver]
Add 3mg. Agarose-immobilized carbohydrate oxidase, prepared as follows from 200 ml of culture, is also added to the flask. To produce this enzyme, mycelium of Polyporus obtusus ATCC #26733 is grown on yeast/malt extract agar slants as follows: yeast extract (3 g), malt extract (3 g). ), agar (20 g), peptone (5 g) and glucose (10 g) into distilled water (1) and adjust the pH to 6.7. Sterilize the medium at 121°C for 15 min. Then adjust the PH to 6.4. Bacteria are inoculated onto this agar slant medium and grown at 25°C for 7 days. The bacteria grown on this slope are then inoculated into yeast/malt extract medium (20 ml of medium in a 125 ml Erlenmeyer flask) prepared as described above (but without the addition of agar).
This bacterium was grown for 9 days at 25°C on a rotary shaker.
Aspirate the medium through #541 Watman paper in a Buchner funnel. The enzyme is contained in mycelium that is kept on paper. The mycelium obtained from 400ml of medium was 0.05N at PH7.0.
Place in a Waring blender containing 70 ml of potassium phosphate buffer and homogenize for 3 minutes. The mixture is centrifuged at 6000 rpm for 20 minutes and the supernatant liquid is separated from the solids by decantation. Add 19 g of polyethylene glycol (molecular weight 4000) to the supernatant liquid in a 500 ml Erlenmeyer flask, and stir the solution for 30 minutes. Then the suspension at 7000rpm
Centrifuge for 20 minutes. Decant the supernatant and discard. Add 15 ml of 0.2M sodium chloride and 15 ml of 0.05M potassium phosphate buffer (PH7.0) to the precipitate and stir. If the solution is allowed to stand for 30 minutes, a precipitate will form during that time. Centrifuge this mixture at 14000 rpm for 20 minutes. The opaque white supernatant containing the cell-free pure enzyme is separated by decanting. Immobilization of this enzyme onto agarose is carried out as follows: pure, cell-free enzyme is added to 500 ml of distilled water.
Dialyze overnight against Then 0.1M at PH8.0
Add 5 ml of sodium bicarbonate. To this solution is added activated CH-Sepharose 4B (washed and reswollen on a sintered glass filter with 500 ml of 1mM HCl). end-over-end mixer (end-over-
Stir the gel suspension for 1 hour at 25°C using an end mixer. The gel suspension was then first washed with 40 ml of 0.1 M sodium bicarbonate (PH8.0) and then
0.05M Tris containing 0.5M Sodium Chloride
Wash with 40 ml of buffer (PH 8.0) and then with 0.5 M sodium formate buffer (PH 4.0) containing 0.5 M sodium chloride. Samples of the reaction mixture were withdrawn whenever any changes occurred and analyzed for glucose and D-glucosone. HPLC is used to quantify the peak areas at Rt 11.5 min (glucose) and Rt 14.0 min (D-glucosone) and calculate the extent of sugar present. The following results were obtained:

[Table] Following the entire course of the reaction, the substantial conversion of glucose to D-glucosone was Rf0.58 (glucose)
The spot disappears and is also indicated by the appearance of the Rf0.43-0.49 (D-glucosone) streak,
Using an in vitro colorimetric assay of TTC, this is indicated by an increase in absorbance at 480 nm over the course of the reaction. That the product is indeed D-glucosone is confirmed by fragmentation followed by mass spectrometry as described above. 512 squares (molecular ion) and 223 squares (OC−CH=
A characteristic D-glucosone mass ion in the N-NO/ ₂ ion fragment) is obtained for this product. EXAMPLE This example demonstrates the use of glucose-2-oxidase to perform essentially complete enzymatic conversion of glucose to D-glucosone. The reaction and conditions of the example (using agarose as immobilization carrier) are repeated using glucose-2-oxidase instead of carbohydrate oxidase. Five hours after starting the reaction, more than 99% of the glucose was converted to D-glucosone. To produce the glucose-2-oxidase enzyme, mycelial cultures of Asbergillus oryzae ATTC #7252 are carried out in bovine, yeast extract/tryptone medium as follows: bovine extract (5 g), yeast extract (5 g), Tryptone (3g), Dextrose (1g) and Difco Starch (24g)
into distilled water (1) and adjust the pH to 7.3.
Sterilize the medium at 121 °C for 35 min. Using spores obtained by a conventional method, a medium is inoculated at an amount of about 3×10 ⁴ spores/ml, and grown at 30° C. for 2 days on a rotary shaker (180 rpm). The culture is aspirated through #541 Watzmann paper using a Buchner Toto and washed several times with water. This enzyme is contained in the mycelium that remains on the paper. Purification and immobilization of this enzyme can proceed using the procedures in the Examples. EXAMPLES In the examples, the level of free hydrogen peroxide produced by the reaction of carbohydrate oxidase and glucose in connection with D-glucosone production was minimized by decomposing the hydrogen peroxide with catalase. Another way to minimize the level of free hydrogen peroxide is to couple this production process to a hydrogen peroxide utilization reaction that produces suitable (useful) by-products. In this example, the production of hydrogen peroxide is coupled to the production of propylene-bromohydrin, which is an intermediate in propylene oxide synthesis according to the theory detailed in the authors' US Patent Application No. 39337 and US Pat. No. 4,247,641. The reaction of treating glucose with immobilized carbohydrate oxidase of Polyporus obtusus ATCC #26733 to produce D-glucosone and hydrogen peroxide was obtained from Coralina sp. in the presence of bromide and propylene. By combining the reaction with immobilized seaweed peroxidase, propylene bromohydrin is produced. The end result of this coupling reaction is then the co-production of glucosone for the production of fructose and propylene bromohydrin, which is easily converted to propylene oxide as described in patent applications No. 39337 and No. 42219. Any enzyme capable of oxidizing the carbon 2 hydroxyl group of glucose in conjunction with the production of hydrogen peroxide can be used for the production of alkene-halohydrins according to the teachings of the authors' earlier references, pending patents. The complex and the peroxidase for halogenation can be combined. Cell-free purified seaweed peroxidase enzyme is produced as follows. Coralina sp., obtained along the La jolla coast of California, is placed in a Virtis 45 homogenizer and ground for 5 minutes in distilled water. Centrifuge the homogenate at 20,000 rpm for 20 minutes. Transfer the supernatant by decanting and save. Resuspend the residual mass in distilled water and centrifuge again. Combine this supernatant with the previous supernatant. This solution is first brought to 33% and then 55% saturation by adding ammonium sulfate.
Centrifugation and clump separation operations are performed at each step. this
The 33% to 55% mass fraction is applied to a DEAE column and gradient elution is performed using 0.3M to 1M phosphate buffer (PH6.0). The fraction eluted at 1M concentration is dialyzed overnight against 20mM phosphate buffer (PH6). Immobilized seaweed peroxidase is prepared as follows: Glass beads (obtained from Sigma Chemical Company, PG-
700−200) in 18 ml of deionized water with 1 g of glass beads.
Activate by suspending. Add 2 ml of 10% (vol/vol) α-aminopropyltriethoxysilane and adjust the PH of the mixture to 3-5 with 6N HCl. Shake the mixture at 75°C for 2 hours. Vacuum dry the glass beads at 80°C overnight. 3.2 ml of purified Coralina sp. enzyme prepared as above.
and 50 mg of water-soluble carbodiimide to the glass beads. Adjust the pH to 4.5 and shake the mixture overnight at 4°C. This product (enzyme coated beads) is washed with water. Its activity is measured as 2 monochlorodimedone units/g of weight of beads. Carbohydrate oxidase immobilized on agarose is prepared from 10 ml of cell-free purified enzyme as in the example. A reaction mixture containing the following components is set up in a 100 ml Pyrex flask: (a) seaweed peroxidase-coated glass beads, 1
g, (b) immobilized carbohydrate oxidase prepared as above, (c) 800 mg of potassium bromide and (d) 20 ml of 0.01M potassium phosphate buffer (PH7.0). The flask is continuously blown with both propylene and oxygen. The reaction is started with 2 gm glucose. After 20 hours, a sample is taken from the reaction solution and analyzed for residual glucose, D-glucosone, and propylene bromohydrin. The produced propylene bromohydrin is analyzed as follows: 5μ of the reaction mixture is placed on a Hewlett-Packard model 402 gas chromatograph with a 6-foot x 1/8 inch glass column and a Porapak R ( 80/100 mesh) into the packed container. The flow rate was adjusted to 30 ml/min for helium gas, and the column temperature was 200°C.
The retention time for propylene bromohydrin was 9 minutes for 1-bromo-2-propanol, and 9 minutes for 2-bromo-1-propanol.
Propanol is 10 minutes. The identity of the product is confirmed by comparison with a standard of propylene bromohydrin: 1-bromo-
2-Propanol was purchased from Pfaltz and Bauer, Inc. 2-Bromo-1-propanol is synthesized by reduction of 1-bromopropionyl chloride with lithium aluminum hydride. The reaction product and the standard have identical retention times and identical mass spectrometry results: Bromine is identified by the presence of M and M+2 isotopic groups of equal intensity: the molecular ion for both isomers is chemically isolated with isobutane reagent gas. confirmed by ionization (M ⁺ ; m/e138
+140); the main fragmentation for 1-bromo-2-propanol is the predicted fragment in which CH ₂ Br is lost, while for 2-bromo-2-propanol
For 1-propanol, the main fragmentation is expected to be the loss of CH ₃ CHBr. Analysis of the sample indicates that the conversion of glucose to D-glucosone is greater than 99% and the propylene bromohydrin production is estimated to be 20 gm/. EXAMPLE This example serves to further explain the concepts presented in the example. In this case, immobilized glucose-2-oxidase is replaced with immobilized carbohydrate oxidase. Immobilized seaweed peroxidase enzyme is produced in the same manner as in the Examples. Immobilized glucose-2-oxidase is produced in the same manner as in the examples. The reaction mixture was the same as in the example, but instead of immobilized carbohydrate oxidase, immobilized glucose-2-
Produced by replacing oxidase. After 20 hours, samples are taken from the reaction and analyzed for residual glucose, D-glucosone, and propylene bromohydrin. The results show that the conversion of glucose to D-glucosone is over 99%, and the production of propylene bromohydrin is 19.5 gm/
It was shown that EXAMPLE This example illustrates the conversion of glucose to D-glucosone in high yields over long reaction times using immobilized carbohydrate oxidase in a column reactor. Carbohydrate oxidizer produced in the same manner as in the example
(cell-free, purified enzyme) (10 ml) is immobilized on hydroxyapatite (calcium hydrogen phosphate) as follows: 100 ml of cell-free purified enzyme and 20 g of hydroxide apatite.
Add 100 ml of 1 mM potassium phosphate buffer (PH7.0) solution. The mixture is stirred for 30 minutes and then the solids are separated from the liquid by decanting. Then, the solid matter was first dissolved in 10mM potassium phosphate buffer (PH7.0) at 200%
ml and then with 200 ml of distilled water. This material was then added to a glass column (0.5 cm x 4.5
cm). A 1% glucose solution is passed through this column at a flow rate of 1.5 ml for 1 hour. The eluate is analyzed periodically for detection of residual glucose and D-glucosone produced. The eluate that was continuously run for 5 days showed that the conversion of glucose to D-glucosone was 95% or more. No hydrogen peroxide was detected.
The study ended before the true half-life of the enzyme could be determined. At slow efflux rates, such as those in this experiment, both the availability of oxygen and the lack of accumulation of hydrogen peroxide are key to substantially complete conversion of glucose to D-glucosone. In this case, the hydroxide apatite, which is the base material of the carrier, decomposed hydrogen peroxide. Reference 1 Specialized Sugars for the Food
Industry, edited by Jeanne C. Johnson,
Noyes Data Corporation, 1976, pp.130−
201. 2 CRBecker and CE May, J.Amer.Chem,
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Claims (1)

[Scope of Claims] 1. An aqueous solution of glucose is prepared, and the glucose in the solution state is substantially completely converted into D-glucosone in the solution state while removing hydrogen peroxide as a by-product. A method for producing D-glucosone comprising converting it. 2. The method according to claim 1, wherein the enzyme is an oxidoreductase having glucose-2-oxidase activity. 3 The enzyme is glucose-2- from Aspergillus oryzae.
Oxidase and pyranose-2- from Polyporus obtusus
3. The method of claim 2, wherein the oxidase is selected from the group consisting of oxidases. 4 Claim 3 in which the enzyme is immobilized
The method described in section. 5. The method according to claim 1, wherein hydrogen peroxide is removed by enzymatic decomposition with catalase. 6. By preparing an aqueous solution of glucose and using an immobilized oxidoreductase enzyme having glucose-2-oxidase activity, the glucose in the solution state is substantially oxidized while removing the by-product hydrogen peroxide. 1. A process for producing D-glucosone, which comprises completely converting D-glucosone into solution-state D-glucosone and recovering D-glucosone in substantially pure form. 7. The method according to claim 6, which is carried out in a column reactor.